Intestinal epithelial cell necrosis is a common pathologic feature of many gastrointestinal disorders including inflammatory bowel disease, infectious gastroenteritis and necrotizing enterocolitis. Although the etiology of intestinal cell necrosis is uncertain, risk factors include anisotonic fluids, toxins, ischemia- reperfusion injury and reactive oxygen species. These stressors perturb peptidase-inhibitor balance, which leads to excessive intracellular proteolysis, loss of membrane integrity, dissolution of sub-cellular architecture and necrotic cell death. The strong association between promiscuous intracellular proteolysis and cell death suggests that peptidase inhibitors serve as necrotic cell death regulators. Extensive searches within the largest family of peptidase inhibitors, the serpins (e.g., antithrombin and 11-antitrypsin), failed to yield candidate regulatory genes. However, this result was not surprising as most serpins are secreted and unlikely to play a regulatory role intracellularly. Recently, we identified a subset of serpins that are abundantly expressed within the cytoplasm of metazoan epithelial cells, including those of the mammalian gastrointestinal tract. Since some of these intracellular serpins (serpinsIC) neutralize lysosomal cysteine and serine peptidases, we hypothesized that they regulate intracellular proteolysis and enhance cell survival. This hypothesis was confirmed by showing that the C. elegans serpinIC, SRP-6, exhibits a pro-survival function by blocking intestinal epithelial cell necrosis. After hypotonic shock, thermal stress, hyperoxia, hypoxia or cation channel hyperactivity;srp-6 nulls underwent a catastrophic series of events culminating in intestinal cell lysosomal disruption, cytoplasmic proteolysis and whole animal death. This newly defined necrotic death phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. SRP-6 provided protection by blocking both the induction of, and the lethal effects from, lysosomal injury. Taken together, we now hypothesize that multiple noxious stimuli converge upon an evolutionarily conserved, peptidase-driven core stress-response pathway that, in the absence of serpinIC regulation, leads to necrotic cell death rather than cell survival. The goal of this proposal is to identify the core molecular components underlying this stress-response pathway by employing powerful unbiased genetic approaches in C. elegans. As a corollary, we will define the intracellular peptidase targets of SRP-6. Finally, comparisons to mammalian systems will determine whether this evolutionarily ancient, serpinIC anti-peptidase defense system regulates intestinal epithelial cell fitness in higher vertebrates. Conservation of this pathway provides the rationale for eventually developing novel anti- necrosis therapeutics using this C. elegans platform. The specific aims are to 1) define the genetic basis of C. elegans intestinal cell necrosis and determine how the stress-response pathway, serpinsIC and lysosomal peptidases determine cell viability, 2) identify the necrotic cell death peptidases regulated by SRP-6 and 3) assess the extent to which mammalian serpinsIC regulate necrotic cell death. PUBLIC HEALTH RELEVANCE: Necrosis is a type of cell death that affects the lining of the intestines and other tissues including the heart and brain. By studying intestinal necrosis in a simpler model system, we will learn how cell death occurs so we can begin to develop new means to treat this very common cause of human disease.